1. Field of the Invention
The present invention relates to novel glucocorticoid receptors that have greater affinity and specifity than the naturally occurring receptors. More particularly, the invention relates to the altering of the equivalent of cysteine-656 in the rat glucocorticoid receptor to either serine or glycine for the production of super glucocorticoid receptors which retain full biological activity in intact cells and have higher affinity and specificity of glucocorticoid steroid binding than the original receptor. The invention further relates to the recombinant expression of such altered receptors in host cells.
2. Background Information
The successes of the last several years in cloning the glucocorticoid receptor from several species (S. M. Hollenberg et al., Nature 318:635-641 (1985); R. Miesfeld et al., Cell 46:389-399 (1986); M. Danielson et al., EMBO J. 5:2513-2522 (1986)) have revealed a dramatic conservation in amino acid sequence in the receptors from human, rat, and mouse. The complete sequence for rat glucocorticoid receptor as set forth in R. Meisfeld et al. (1986) is shown in SEQ ID NO. 5. The amino acid sequences of human and rat receptors are 88% identical; this value rises to 92% if one allows for conservative amino acid differences. Likewise the human receptor was found to be 89% homologous with the mouse receptor (Danielson et al., (1986)). Given the extremely high homology between these glucocorticoid receptors, it is not surprising that the human and rat receptors "appear to be operationally indistinguishable" (Miesfeld et al., (1986)).
Steroid binding is the first step in a series of events that translate the structural information of the steroid into the observed biological response. Molecular biology experiments have defined the 250 carboxyl terminal amino acids as being the steroid binding domain of the receptor (V. Giguere et al., Cell 46:645-652 (1986); S. Rusconi and K. R. Yamamoto, EMBO J. 6:1309-1315 (1987)). In this region, &gt;96% of the amino acid sequence in the human, rat, and mouse receptors is identical. Thus it is not unexpected that the affinity of various steroids for human, rat, and mouse glucocorticoid receptors has been found to be almost identical.
There are separate receptor proteins for each of the five classical steroid hormones--androgen, estrogen, glucocorticoid, mineralocorticoid, and progesterone. Each of these receptors exhibits both a common structural organization of functional domains and appreciable homology between the steroid-binding domains (R. M. Evans, Science 240: 889-895 (1988)). This homology offers a reasonable explanation for the fact that virtually all steroids appear to interact with more than one class of receptors (J. -P. Raynaud and T. Ojasoo, In Steroid Hormone Receptors: Structure and Function, (H. Eriksson, and J. -A. Gustafsson, eds.), 141:170 (1983); G. Teutsch et al., J. Steroid Biochem 31:549-565 (1988)).
It is possible to achieve total selectivity among the five classical steroid receptors (androgen, estrogen, glucocorticoid, mineralocorticoid, and progesterone) with anti-receptor antibodies. However, selective recognition of the biologically active form of receptors via the binding of specific ligands has been elusive (Raynaud and Ojasoo, 1983; Teutsch et al., 1988). The consequences of such cross-reactivity are manifold. It complicates the identification of the steroid binding form of receptors (S. Lopez et al., J. Biol. Chem. 265:16039-16042 (1990)) and causes unwanted side effects in vitro experiments with cells containing the offending receptors. In clinical settings, the side effects can be very severe, such as to limit long term glucocorticoid therapy to only those cases that are not easily remedied by other protocols (G. H. Williams and R. G. Dluhy, In "Harrison's Principles of Internal Medicine." 11th ed. (Braunwald, E., Isselbacher, K. J., Petersdorf, R. G., Wilson, J. D., Martin, J. B., and Fauci, A. S., eds) McGraw-Hill, New York, pp. 1772-1774 (1987)).
One solution to this problem is to use molecular biology to modify the steroid binding domain of the glucocorticoid receptor. An increase in the specificity of steroid binding would be one desirable change. Another would be to increase the affinity of steroid binding, since lowering the concentrations of steroid needed for full glucocorticoid response would also decrease the binding (and lower the biological responses) with other receptors. However, the published reports indicate that this will be very difficult to accomplish. Terminal deletions to give species smaller than amino acids 497-795 (all numbering is for the rat receptor sequence) resulted in more than a 300 fold reduction in affinity (Rusconi and Yamamoto, 1987). Similarly, most internal deletions or substitutions and point mutants eliminated or greatly decreased steroid binding (Giguere et al., 1986; Danielson et al., 1986). It thus appears that, aside from the few changes that are seen in rat vs human vs mouse receptors, the native sequence represents the optimal sequence for binding glucocorticoid steroids with high affinity and specificity. A 16 kDa steroid binding fragment of the rat glucocorticoid receptor has recently been identified (S. S. Simons et al., J. Biol. Chem. 264:14493-14497 (1989)). While this fragment retains the specificity of the intact 98 kDa receptor, and thus can be considered the core of the steroid binding domain, the affinity of this core fragment for glucocorticoid steroids is reduced by about a factor of 23 (Simons et al., 1989). Thus it would be predicted that almost any amino acid change in the 16 kDa core fragment would give rise to a loss of steroid binding, especially if the mutation was of a "crucial" amino acid.
There have been many efforts to identify the "crucial" amino acids involved in steroid binding to the glucocorticoid receptor. The initial candidates were cysteine (J. D. Baxter and G. M. Tomkins, Proc. Natl. Acad. Sci. USA 68: 932-937 (1971)) and lysine and arginine (D. M. DiSorbo et al., Endocrinol 106:922-929 (1980)). In fact, it has been known for many years that intact thiols are involved in the steroid binding of all receptors (R. J. B. King and W. I. P Mainwaring, "Steroid-Cell Interactions", University Park Press, Baltimore (1974)). Direct support for this conclusion was obtained when dexamethasone mesylate (Dex-Mes), a thiol-specific (S. S. Simons, J. Biol. Chem. 262:9669-9675 (1987)) affinity label for glucocorticoid receptors, was shown to covalently label just one thiol in the receptor, i.e., cysteine 656 (Simons et al., 1987). The identical cysteine in the mouse glucocorticoid receptor is also labeled by Dex-Mes (L. I. Smith et al., Biochemistry 27:3747-3753 (1988)). However, recent data indicate that at least 2 thiols are involved in steroid binding. In particular, the formation of an intramolecular disulfide in the steroid-free receptor molecule blocks steroid binding (N. R. Miller and S. S. Simons, J. Biol. Chem. 263:15217-15225 (1988); P. K. Chakraborti et al., Endocrinology 12:2530-2539 (1990)) and preincubation of unbound receptors with arsenite, which selectively reacts with closely spaced or vicinal dithiols, eliminates all steroid binding (S. S. Simons et al., J. Biol. Chem. 265:1938-1945 (1990); Lopez et al., 1990; Chakraborti et al., (1990)). Both thiols were found to be present in the 16 kDa core binding fragment (Chakraborti et al., 1990), which contains only 3 cysteines (Cys-640, -656, and -661). Recent results have identified the vicinal dithiols as being Cys-656 and -661. Thus these two thiols, and possibly Cys-640, would appear to be crucial for the high affinity and specificity of steroid binding to glucocorticoid receptors. Support for this conclusion comes from the facts that no other receptor has the same spacing of closely spaced thiols in the steroid binding domain and that arsenite, which selectively reacts with such closely spaced thiols, blocks the steroid binding activity of glucocorticoid receptors but not of any other steroid receptors (Lopez et al., 1990).
Based on the above information, it would be predicted that mutation of either Cys-656 or Cys-661 would both eliminate, or seriously reduce the affinity of, steroid binding to the glucocorticoid receptor and reduce the specificity of binding. As expected, cysteine-to-serine point mutations of Cys-640 and Cys-661 did cause a 3-4 fold reduction in affinity. However, the same mutation of Cys-656 caused a 3 fold increase in affinity. Even more unexpected was that the cysteine-to-glycine mutation of Cys-656 caused a greater increase in affinity that was accompanied by an increase in the specificity of steroid binding.
The present invention relates, in part, to novel glucocorticoid receptors with cysteine-to-glycine or -serine point mutations at the equivalent position of 656 in the rat receptor. Such super receptors represent improvements on nature that could not have been predicted based on previous work in the field. The present invention further relates to recombinant super glucocorticol receptors with an altered cysteine-to-glycine or serine change at the equivalent position of 656 in the rat receptor expressed in host cells.